Significance of the proposed research. Diarrheal disease caused by enteric bacteria is an important endemic health threat and a major source of food-borne disease. Diarrheal pathogens also remain an important health concern in current military operations and in bioterrorism. In spite of the appreciated magnitude of the threat to both general human health and the severe inhibition of military performance caused by ETEC, we still do not have an effective vaccine, nor do we fully understand the virulence determinants responsible for intestinal colonization and subsequent diarrheal disease. Basic research is needed to identify new therapeutic and vaccine targets. The objective of this application is to use quantitative proteomic protein profiling to discover broadly conserved antigens that will permit development of a vaccine efficacious against ETEC. The primary need that motivates this research objective is that despite the significant effort and expense that has been employed in vaccine trials using ETEC adhesins, none of these vaccines have been especially effective in mediating cross- protective immunity, and some trials have even had significant health risks. Alternative vaccine targets and strategies are demanded. We will utilize our proven experience in ETEC microbiology and quantitative proteomics to characterize novel ETEC vaccine antigens. We will do this by identifying novel surface-exposed ETEC proteins likely to be recognized by the host immune system. We show in preliminary studies that the proposed approach rapidly and economically identifies conserved, surface-exposed ETEC proteins, independent of strain-specific CFs, which can be purified and used in protection assays in a robust animal model of ETEC virulence. The specific aims are to: 1) Characterize ETEC outer membrane proteomes using quantitative proteomic profiling, 2) Produce recombinant ETEC proteins and antisera, and 3) Immunize mice with recombinant ETEC antigens and quantify their ability to protect against an otherwise lethal challenge with ETEC. The proposed research is significant and innovative because it introduces a novel strategy for protective antigen discovery for a neglected pathogen responsible for causing an enormous global diarrheal disease burden. Indeed, we have already discovered seventeen surface-exposed antigens that are potential components of a new ETEC vaccine formulation. Additionally, these experiments will optimize proteomic strategies needed to characterize vaccine candidates in other enteric pathogens.